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BIOSYNTHESIS AND CHARACTERIZATION
OF POLY(3-HYDROXYBUTYRATE-co-4-
HYDROXYBUTYRATE) COPOLYMER
FROM Cupriavidus sp. USMAHM13
HEMA RAMACHANDRAN
UNIVERSITI SAINS MALAYSIA
2013
BIOSYNTHESIS AND CHARACTERIZATION
OF POLY(3-HYDROXYBUTYRATE-co-4-
HYDROXYBUTYRATE) COPOLYMER
FROM Cupriavidus sp. USMAHM13
by
HEMA RAMACHANDRAN
Thesis submitted in fulfillment of the requirements
for the Degree of Doctor of Philosophy
November 2013
ii
ACKNOWLEDGEMENTS
First and foremost, my profoundest gratitude to my supervisor, Assoc. Prof.
Dr. Amirul Al-Ashraf Abdullah whose sincerity and steadfast encouragement had
been my inspiration as I hurdled all the obstacles in the completion of this research
work. I have been extremely lucky to have a supervisor who had supported me
throughout my thesis with his patience and knowledge whilst giving me enough
freedom to work in my own way and responded to my questions so promptly. His in-
depth knowledge on a broad spectrum of microbial physiology and biopolymer field
had been extremely beneficial for me. What I have learnt from him is not just how to
do research and write thesis to meet the graduate requirement but also how to view
this world from a new perspective. I could not wish for a better or friendlier
supervisor. There are no words that can truly express the level of gratitude and
appreciation that I have for him. Thank you Dr.!
It is my pleasure to thank Kak Syairah and Kak Solehah who had helped me
to find my smile whenever I face difficulties in my project. I am ever so grateful for
the numerous ways they had supported me throughout my project and personal life.
My sincere thanks also goes to the other lab mates; Shantini, Rennukka, Kak
Hemalatha, Kak Vigneswari, Kak Muzaiyanah, Kak Faezah, Kak Syifa and Hezreen
for the stimulating discussion and for all the fun we had in the last four years. Special
thanks to Shantini and Rennukka who often had to bear the brunt of my frustration
and rages against the failed experiments. In my daily work, I have been blessed with
a friendly and cheerful junior labmates; Kai Hee, Azura, Izzaty, Sheeda, Azuraini
and Syafirah who had helped me to regain some sorts of healthy mind.
iii
I would like to thank Mr. Segaran, Mr. Zahari, Mr. Johari, Ms. Nurul, Mr.
Muthalib and Ms. Jamilah for assisting me in handling various equipments
throughout my study. My deepest appreciation goes to my friends; Renuga, Gayathri,
Divani and Jeyanthi for their love and unconditional support through the storms of
my life. I would also like to acknowledge the USM Fellowship for funding me
throughout my research.
I am truly indebted and thankful to my family members for their constant
love, support and encouragement through every endeavor, no matter how big or
small. I would not have made it this far without them. I know how much my parents
had sacrificed to give me the best opportunities available and for that I am eternally
grateful. Words alone cannot express my mother’s unending patience, unconditional
love and inner strength that inspires and pushes me to constantly do better. I also
know that I can always count on my siblings for advice, undying support and
laughter which always make me feel lighter and stronger.
My sincerest gratefulness goes to Mr. Harinderan who always make me feels
loved. I know I can do anything because he will always have my back as he had
proven it for the past eight years. He had been there whenever I need someone to
lean on through all of life’s trials. He had helped me to remain stable in times of
instability and guided me in my moments of confusion. Though no amount of "thank
you" will suffice, I wanted him to know that I appreciate the varieties of support that
he had given me whether emotional, informational or tangible.
Last but not least, I owe my gratefulness to God for answering my prayers by
giving me the strength and perseverance to complete my doctoral study successfully.
Thank you so much Dear Lord.
iv
TABLE OF CONTENTS
PAGE
ACKNOWLEDGEMENTS ..................................................................................... ii
TABLE OF CONTENTS ....................................................................................... iv
LIST OF TABLES .................................................................................................. x
LIST OF FIGURES……………………………………………………………... xiii
LIST OF SYMBOLS AND ABBREVIATIONS ................................................. xv
ABSTRAK .............................................................................................................. xx
ABSTRACT ......................................................................................................... xxii
1.0 INTRODUCTION...................................................................................... 1
1.1 Problem statements…………………………………………………….. 4
1.2 Objectives………………………………………………………………. 5
2.0 LITERATURE REVIEW .......................................................................... 6
2.1 Polyhydroxyalkanoates: The prospect green biopolymer ............................. 6
2.1.1 History of PHAs ................................................................................ 8
2.1.2 Types of PHAs and their physical properties ................................... 10
2.1.2.1 Short-chain-length (SCL)-PHAs ........................................... 10
2.1.2.2 Medium-chain-length (MCL)-PHAs .................................... 12
2.1.2.3 Short-chain-length-medium-chain-length (SCL-MCL)- ...... 14
PHAs
2.1.3 Biosynthesis of PHAs ....................................................................... 16
2.1.3.1 Biosynthesis of SCL-PHAs .................................................. 16
2.1.3.2 Biosynthesis of MCL-PHAs ................................................. 17
v
2.2 Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] ............ 19
2.2.1 Biosynthesis of P(3HB-co-4HB) copolymer by various .................. 19
microorganisms
2.2.2 Biosynthetic pathway of P(3HB-co-4HB) copolymer ...................... 26
2.2.2.1 Biosynthesis of P(3HB-co-4HB) copolymer using ............... 26
carbon precursor
2.2.2.2 Biosynthesis of P(3HB-co-4HB) copolymer using ............... 27
unrelated carbon source
2.2.3 Structure and properties of P(3HB-co-4HB) copolymer .................. 29
2.2.3.1 X-ray crystallinities ............................................................... 29
2.2.3.2 Molecular mass ..................................................................... 30
2.2.3.3 Mechanical properties ........................................................... 32
2.2.3.4 Thermal properties ................................................................ 34
2.2.3.5 Biodegradation ..................................................................... 35
2.2.3.6 Biocompatibility ................................................................... 38
2.3 Glycerine: A promising and renewable carbon source ................................. 40
2.3.1 Biosynthesis of PHAs using glycerine .............................................. 43
2.3.2 Production and treatment of glycerine pitch from oleochemicals .... 47
industry in Malaysia
2.4 Concluding remark ....................................................................... 52
3.0 MATERIALS AND METHODS .............................................................. 54
3.1 Carbon sources .............................................................................................. 54
3.2 Sterilization method ...................................................................................... 54
3.3 Determination of bacterial growth (cell dry weight) .................................... 54
3.4 Centrifugation of culture ............................................................................... 55
vi
3.5 Medium ......................................................................................................... 55
3.5.1 Bacterial growth medium .................................................................. 55
3.5.2 PHA production medium .................................................................. 56
3.6 Isolation and Nile red screening of P(3HB-co-4HB)-accumulating ............. 57
bacteria
3.7 Screening of P(3HB-co-4HB)-accumulating bacteria through gas .............. 57
chromatography (GC) analysis
3.8 Bacterial strain and maintenance .................................................................. 58
3.9 Characterization of Cupriavidus sp. USMAHM13 ...................................... 58
3.9.1 Morphological characterization ........................................................ 58
3.9.1.1 Colony characterization ...................................................... 58
3.9.1.2 Cell characterization ........................................................... 59
3.9.2 Gram-staining ................................................................................... 59
3.9.3 Physiological and biochemical characterization ............................... 59
3.9.3.1 Oxidase ............................................................................... 59
3.9.3.2 Catalase ............................................................................... 59
3.9.3.3 Optimum temperature ......................................................... 60
3.9.3.4 Biochemical test (API 20NE KIT) ...................................... 60
3.9.3.5 Biochemical test (Biolog GEN III) ..................................... 60
3.9.3.6 Lipase test ........................................................................... 60
3.9.4 Molecular and chemotaxonomy characterization ............................. 61
3.10 Biosynthesis of P(3HB) and P(3HB-co-4HB) polymer using various .......... 61
carbon sources by Cupriavidus sp. USMAHM13
3.10.1 One-stage cultivation ........................................................................ 61
3.10.2 Two-stage cultivation ........................................................................ 62
vii
3.10.3 Effect of different combinations of carbon sources .......................... 63
3.10.4 Effect of different batches of glycerine pitch ................................... 63
3.10.5 Effect of fructose and glycerine pitch (GPB) on PHA ..................... 64
accumulation in bioreactor and polymer characterization
3.11 Biosynthesis of P(3HB-co-4HB) copolymer using glycerine pitch (GPB) .. 65
and 1,4-butanediol
3.11.1 Effect of different nitrogen sources .................................................. 65
3.11.2 Effect of different concentrations of glycerine pitch and ................. 65
1,4-butanediol
3.11.3 Effect of different concentrations of ammonium acetate .................. 66
3.11.4 Effect of recovered components of glycerine pitch .......................... 66
3.11.5 Effect of ammonium acetate using different batches of .................... 67
glycerine pitch
3.12 Optimization of P(3HB-co-4HB) copolymer production using .................... 68
response surface methodology (RSM)
3.12.1 Central composite design (CCD) ...................................................... 68
3.12.2 3D surface and ANOVA ................................................................... 69
3.13 Biosynthesis of P(3HB) and P(3HB-co-4HB) polymer via batch ................ 70
fermentation in bioreactor
3.14 Characterization of polymer films ................................................................ 71
3.14.1 Molecular mass ................................................................................. 71
3.14.2 Nuclear magnetic resonance (NMR) analysis ................................... 72
3.14.3 Mechanical properties ....................................................................... 73
3.14.4 Thermal properties ............................................................................ 73
3.15 Analytical procedures ................................................................................... 74
viii
3.15.1 Cell dry weight determination .......................................................... 74
3.15.2 Preparation of methanolysis solution ................................................ 74
3.15.3 Preparation of caprylic methyl ester (CME) solution ....................... 74
3.15.4 Methanolysis ..................................................................................... 75
3.15.5 Gas chromatography analysis ........................................................... 76
3.15.6 Enumeration method ......................................................................... 76
3.15.7 Tukey test .......................................................................................... 78
3.15.8 Recovery of various components of glycerine pitch ......................... 78
3.15.9 KLa determination using fermentative dynamic method .................. 78
3.15.10 Polymer film casting…………………………................................. 79
3.16 Microscopic observation .................................................................. 80
3.16.1 Phase contrast microscopy ................................................................ 80
3.16.2 Fluorescence microscopy .................................................................. 80
3.16.3 Scanning electron microscope (SEM) .............................................. 81
3.16.4 Transmission electron microscope (TEM) ........................................ 81
3.16.4.1 Fixation of samples ............................................................. 82
3.16.4.2 Sectioning of the resin blocks ............................................. 83
3.17 Overview of research methodology………………………………………... 84
4.0 RESULTS AND DISCUSSION ................................................................. 86
4.1 Isolation and screening of P(3HB-co-4HB) producers from Malaysian ...... 86
environment
4.2 Biochemical and molecular identification of the isolate USMAHM13 ...... 96
4.3 Biosynthesis of P(3HB-co-4HB) copolymer using various carbon ............ 119
sources by Cupriavidus sp. USMAHM13
ix
4.4 Biosynthesis of P(3HB-co-4HB) copolymer using glycerine pitch (GPB).. 141
and 1,4-butanediol by Cupriavidus sp. USMAHM13
4.5 Optimization of P(3HB-co-4HB) copolymer production using ................. 154
response surface methodology (RSM)
4.6 Biosynthesis and characterization of P(3HB) and P(3HB-co-4HB) .......... 169
polymer via batch fermentation using bioreactor
5.0 CONCLUSION ........................................................................................ 193
5.1 Summary .................................................................................................... 193
5.2 Limitations and recommendations for future work ................................... 195
REFERENCES ................................................................................................... 197
APPENDICES
LIST OF PUBLICATIONS
x
LIST OF TABLES
PAGE
Table 2.1 The chemical structure of common PHAs 15
Table 2.2 Thermal and mechanical properties of the P(3HB-co-4HB) 36 copolymers with various 4HB monomer compositions
Table 2.3 Comparison of glycerine pitch, crude glycerine and purified 51
glycerine from waste of palm oil based biodiesel plant with commercial glycerine
Table 3.1 Compositions of nutrient rich (NR) medium 55
Table 3.2 Compositions of mineral salts medium (MSM) 56
Table 3.3 Compositions of trace elements dissolved in 1 l o f 56 hydrochloric acid (HCl) 0.1 M
Table 3.4: Range of variables at different levels for the centra l 68
composite design Table 3.5 Medium compositions for each experiment carried out in 70
bioreactor Table 3.6 The essential guidelines of Shimadzu GC-2014 operation 76 Table 4.1 Isolation of P(3HB-co-4HB) producers from Perak, 87
M a l a y s i a u s i n g s e l e c t i v e m e d i u m c o n t a i n i n g γ-butyrolactone as carbon source
Table 4.2 Screening of P(3HB-co-4HB) producers using selective 90
medium containing γ-butyrolactone and Nile Red
Table 4.3 Quantitative screening of P(3HB-co-4HB) producers using 92 gas chromatography (GC) analysis
Table 4.4 Morphological characteristics of the isolate USMAHM13 98 Table 4.5 Physiological characteristics of the isolate USMAHM13 101 Table 4.6 BIOLOG metabolic profiles of the isolate USMAHM13 102 Table 4.6 Continued 103
Table 4.7 Partial 16S rRNA gene sequence of the Cupriavidus sp. 105 USMAHM13
xi
Table 4.8 Top five hits of similarity search with BLAST 106
Table 4.9 Complete 16S rRNA gene sequence of the Cupriavidus sp. 108 USMAHM13
Table 4.10 RiboPrinter microbial characterization of Cupriavidus sp. 112 USMAHM13, Cupriavidus sp. USMAA1020 and
Cupriavidus sp. USMAA2-4
Table 4.11 DNA-DNA hybr idizat ion of the Cupriavidus sp . 114 USMAHM13 against Cupriavidus sp. USMAA1020 and Cupriavidus sp. USMAA2-4
Table 4.12 Cellular fatty acids profile of the Cupriavidus sp. USMAHM13 116
comparing to the nearest phylogenetic strains in the genus Cupriavidus
Table 4.13 Biosynthesis of P(3HB) homopolymer using various 120
carbon sources by Cupriavidus sp. USMAHM13
Table 4.14 Biosynthesis of P(3HB-co-4HB) copolymer using different 125
carbon precursors by Cupriavidus sp. USMAHM13
Table 4.15 Biosynthesis of P(3HB-co-4HB) copolymer using different 129 combinations of carbon sources by Cupriavidus sp. USMAHM13
Table 4.16 Biosynthesis of P(3HB-co-4HB) copolymer using different 132 batches of glycerine pitch by Cupriavidus sp. USMAHM13
Table 4.17 Compositions of two different batches of glycerine pitch 133 Table 4.18 Fatty acid compositions of two diffe rent batches of 134
glycerine pitch Table 4.19 Molecular weight and mechanical properties of the polymers 139 Table 4.20 Effect of different nitrogen sources on the biosynthesis of 142
P(3HB-co-4HB) copolymer using combination of glycerine pitch and 1,4-butanediol
Table 4.21 Effect of different concentrations of glycerine pitch and 146
1,4-butanediol on the biosynthesis of P(3HB-co-4HB) copolymer
Table 4.22 Effect of different concentrations of ammonium acetate on 148
the biosynthesis of P(3HB-co-4HB) copolymer using combination of glycerine pitch and 1,4-butanediol
xii
Table 4.23 Effect of recovered components of glycerine pitch on the 150 biosynthesis of P(3HB) and P(3HB-co-4HB) polymer
Table 4.24 Effect of ammonium acetate on the biosynthesis of P(3HB) 153 and P(3HB-co-4HB) polymer using different batches of glycerine pitch
Table 4.25 Experimental design for medium optimizat ion o f 155
P(3HB-co-4HB) copolymer production as given by response surface methodology
Table 4.26 Analysis of variance and regression for cell dry weight 157 Table 4.27 Analysis of variance and regression for PHA content 158
Table 4.28 Analysis of variance and regression for 4HB monomer 160
composition
Table 4.29 Verification of the model using optimized condition given 168 by the software for the maximized P(3HB-co-4HB) copolymer production
Table 4.30 Main characteristics in batch fermentation of P(3HB) and 171 P(3HB-co-4HB) polymer by Cupriavidus sp. USMAHM13 under various conditions
Table 4.31 Molecular weight and dyad sequence distribution of P(3HB) 184 and P(3HB-co-4HB) polymer films
Table 4.32 Mechanical and thermal properties of P(3HB) and 187
P(3HB-co-4HB) polymer films
Table 4.33 Characteristic comparison between pigmented and 192 non-pigmented polymer films
xiii
LIST OF FIGURES
PAGE
Figure 2.1 Biosynthetic pathways of short-chain-length (SCL)-PHA, 18
medium-chain-length (MCL)-PHA and short-medium-chain-
length (SCL-MCL)-PHA from sugars and oils
Figure 2.2 Sources of 4-hydroxybutyryl-CoA for biosynthesis of PHA 28
containing 4HB as constituent
Figure 2.3 Flow diagram of transesterification leading to generation of 49
glycerine pitch in a palm kernel methyl ester plant
Figure 4.1 Observation of PHA-producing microorganisms under UV 91
light which emitted pink fluorescence in the presence of
PHA
Figure 4.2 Microscopic observation of the isolate USMAHM13 94
containing 42 wt% of PHA cultured in MSM containing
γ-butyrolactone as sole carbon source for 72 hours through
two-stage cultivation
Figure 4.3 Morphological characterization of the isolate USMAHM13 97 Figure 4.4 16S rRNA gene sequence similarity of the Cupriavidus 110
sp. USMAHM13 and related taxa
Figure 4.5 Neighbour-joining tree based on 16S rRNA gene sequences 111
showing the position of Cupriavidus sp. USMAHM13
among its phylogenetic neighbours
Figure 4.6 Biosynthesis of P(3HB) homopolymer using different 137
carbon sources in 3.6 l bioreactor through batch
fermentation for 72 hours at 30°C with agitation speed of
200 rpm and 0.4 vvm
Figure 4.7 Biosynthesis of P(3HB-co-4HB) copolymer using different 138
carbon sources with addition of 1,4-butanediol (5 g/l) in
3.6 l bioreactor through batch fermentation for 72 hours at
30°C with agitation speed of 200 rpm and 0.4 vv m
Figure 4.8 Metabolic pathway of P(3HB-co-4HB) copolymer 143
synthesis by Cupriavidus sp. USMAHM13
xiv
Figure 4.9 3D response surface towards cell dry weight 161 Figure 4.10
3D response surface towards PHA content
163
Figure 4.11 3D response surface towards 4HB monomer composition 165 Figure 4.12 Biosynthesis of P(3HB) and P(3HB-co-4HB) polymers 170
through batch fermentation in 3.6 l bioreactor using
different medium compositions
Figure 4.13 Bacterial growth profile of Cupriavidus sp. USMAHM13 172
for the seven experiments conducted through batch
fermentation using 3.6 l bioreactor
Figure 4.14 Time profile of PHA accumulation by Cupriavidus sp. 174
USMAHM13 for the seven experiments conducted through
batch fermentation using 3.6 l bioreactor
Figure 4.15 Time pro f i l e o f 4HB monomer accumulat ion b y 177
Cupriavidus sp. USMAHM13 for the seven experiments
conducted through batch fermentation using 3.6 l bioreactor
Figure 4.16 Dissolved oxygen (DO) profile of seven experiments 181
o b t a i n ed f r o m t h e b i o s yn t h e s i s o f P ( 3 H B) an d
P(3HB-co-4HB) polymers through batch fermentation using 3.6 l bioreactor
Figure 4.17 P(3HB) and P(3HB-co-4HB) polymer films with various 191
4HB monomer compositions produced by Cupriavidus sp.
USMAHM13
xv
LIST OF SYMBOLS AND ABBREVIATIONS
Symbols and Abbreviations Full name
% Percentage
β Beta
γ Gamma
°C Degree Celsius
ΔHm Heat of fusion
g Gravity
CL Dissolved oxygen concentration
C*L Dissolved oxygen concentrations in equilibrium with mean gaseous oxygen concentration
Da Dalton
g Gram
g/l Gram per liter
J/g Joule per gram
kDa KiloDalton
kg Kilogram
KLa Volumetric oxygen transfer coefficient
l Liter
M Molar
Mn Number-average molecular weight
Mw Average molecular weight
Mw/Mn Polydispersity index
mg Milligram
mg/ml Milligram per milliliter
ml Milliliter
xvi
mm Millimeter
mmol/l Millimole per liter
mM Millimolar
Mol% Mole percentage
MPa Mega Pascal
nm Nanometer
ppm Parts per million
psi Pounds per square inch
QO2X Oxygen uptake rate of the cells
R Correlation coefficient
R2 Determination coefficient
rcf Rotation centrifugational force
rpm Rotation per minute
Tg Glass transition temperature
Tm Melting temperature
Tc Crystallization temperature
µg Microgram
µg/ml Microgram per milliliter
µl Microliter
µm Micrometer
v/v Volume per volume
wt% Weight percent
w/v Weight per volume
w/w Weight per weight
3HB 3-hydroxybutyrate
3HB-CoA 3-hydroxybutyryl-CoA
4HB 4-hydroxybutyrate
xvii
4HB-CoA 4-hydroxybutyryl-CoA
ACP Acyl carrier protein
ANOVA Analysis of variance
ASTM American society for testing and materials
ATCC American type culture collection
BLAST Basic local alignment search tool
C/N Carbon-to-nitrogen ratio
CaCl2·2H2O Calcium (II) chloride dihydrate
CCD Central composite design
CDCl3 Deuterated chloroform
CDW Cell dry weight
CME Caprylic methyl ester
CoA CoenzymeA
CoCl2·6H2O Cobalt (II) chloride hexahydrate
CoSO4·7H2O Cobalt sulphate heptahydrate
CPKO Crude palm kernel oil
CPO Crude palm oil
CuCl2·2H2O Copper (II) chloride dihydrate
DO Dissolved oxygen
DSC Differential scanning calorimeter
DSMZ Deutsche sammlung von mikroorganismen und zellkulturen
EMBL European molecular biology laboratory
FabG 3-ketoacyl-CoA reductase
FeSO4·7H2O Iron (II) sulphate heptahydrate
FID Flame ionization detector
GC Gas chromatography
xviii
GP Glycerine pitch
GPC Gel permeation chromatography
HA Hydroxyalkanoate
HCl Hydrochloric acid
H2SO4 Sulphuric acid
HMDS Hexamethyldisilazane
ICI Imperial chemical industries
IS Internal standard
KH2PO4 Potassium dihydrogen phosphate
MCL Medium-chain-length
MgSO4·7H2O Magnesium sulphate heptahydrate
MIC Minimal inhibitory concentration
MnCl2·4H2O Manganese (II) chloride tetrahydrate
MSM Mineral salts medium
NA Nutrient agar
NaCl Sodium chloride
Na2SO4 Sodium sulphate
NADH Nicotinamide adenine dinucleotide
NADPH Nicotinamide adenine dinucleotide phosphate
NaOH Sodium hydroxide
NCBI National center for biotechnology information
NH4Cl Ammonium chloride
(NH4)2SO4 Ammonium sulphate
NMR Nuclear magnetic resonance
NR Nutrient rich
OD Optical density
xix
OTR Oxygen transfer rate
OUR Oxygen uptake rate
P(3HB) Poly(3-hydroxybutyrate)
P(3HB-co-3HV) Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)
P(3HB-co-4HB) Poly(3-hydroxybutyrate-co-4-hydroxybutyrate)
PDI Polydispersity index
PHAs Polyhydroxyalkanoates
PhaA β-ketothiolase
PhaB NADPH-dependent acetoacetyl-CoA dehydrogenase
PhaC PHA synthase
PhaG 3-hydroxyacyl-ACP-CoA transferase
PhaJ Enoyl-CoA hydratase
PCR Polymerase chain reaction
PO Palm olein
RDP Ribosomal database project
rRNA Ribosomal ribonucleic acid
RSM Response surface methodology
SCL Short-chain-length
SEM Scanning electron microscope
TCA Tricarboxylic acid
TEM Transmission electron microscope
TMS Tetramethylsilane
UV-Vis Ultraviolet-Visible
ZnSO4·7H2O Zinc sulphate heptahydrate
xx
BIOSINTESIS DAN PENCIRIAN KOPOLIMER POLI(3-
HIDROKSIBUTIRAT-ko-4-HIDROKSIBUTIRAT) DARIPADA
Cupriavidus sp. USMAHM13
ABSTRAK
Penghasilan poli(3-hidroksibutirat-ko-4-hidroksibutirat) [P(3HB-ko-4HB)]
dengan menggunakan kombinasi gliserin buangan dan karbon pelopor daripada
bakteria adalah masih sangat terhad. Oleh sebab itu, kajian ini dijalankan untuk (i)
memencilkan dan mengenalpasti bakterium yang berupaya menghasilkan kopolimer
P(3HB-ko-4HB) dengan kandungan PHA dan komposisi monomer 4HB yang tinggi,
(ii) meneroka keupayaan bakterium tersebut dalam menghasilkan kopolimer P(3HB-
ko-4HB) dengan menggunakan pelbagai karbon yang boleh diperbaharui dan murah,
(iii) mengoptimumkan penghasilan kopolimer P(3HB-ko-4HB) dengan
menggunakan tar gliserin melalui fermentasi kelalang goncangan dengan
menggunakan metodologi permukaan respon (RSM) dan (iv) menilai sifat-sifat
bahan kopolimer P(3HB-ko-4HB) dengan pelbagai komposisi monomer 4HB. Dalam
kajian ini, suatu bakteria novel berpigmen kuning yang mempamerkan keupayaan
untuk menghasilkan kopolimer P(3HB-ko-4HB) telah dipencilkan dengan jayanya
dari Perak, Malaysia dan telah dilabelkan sebagai Cupriavidus sp. USMAHM13.
Berdasarkan pada analisis fenotip dan genotip, ia boleh dicadangkan bahawa
Cupriavidus sp. USMAHM13 mewakili suatu spesis novel dalam genus Cupriavidus.
Penyaringan awal substrat karbon untuk penghasilan kopolimer P(3HB-ko-4HB)
oleh Cupriavidus sp. USMAHM13 telah mendedahkan bahawa komposisi 4HB
monomer yang lebih tinggi (43 mol%) dengan berat kering sel dan kandungan PHA
sebanyak 6.0 g/l dan 49% (b/b), masing-masing dicapai melalui kombinasi tar
xxi
gliserin (5 g/l) dan 1,4-butanadiol (5 g/l) secara pengkulturan satu peringkat. Kajian
ini juga mendedahkan bahawa gliserin mentah yang diasingkan daripada tar gliserin
paling menyumbang kepada sintesis kopolimer P(3HB-ko-4HB) dibandingkan
dengan komponen lain yang diasingkan. Peningkatan pengumpulan monomer 4HB
juga dicapai melalui penambahan ammonium asetat sebagai sumber nitrogen yang
bertindak sebagai perangsang 4HB. Pengoptimuman medium dengan menggunakan
RSM melalui fermentasi kelalang goncangan telah menjurus kepada pengumpulan
tertinggi monomer 4HB (51 mol%) dengan berat kering sel dan kandungan PHA
sebanyak 10.1 g/l dan 53% (b/b), masing-masing dengan menggunakan kombinasi
tar gliserin (10 g/l), 1,4-butanadiol (8.14 g/l) dan ammonium asetat (2.39 g/l).
Biosintesis kopolimer P(3HB-ko-4HB) dengan komposisi monomer 4HB berjulat
daripada 3 mol% kepada 40 mol% juga dicapai melalui fermentasi berkelompok
dalam bioreaktor dengan memanipulasi kepekatan ammonium asetat. Kopolimer-
kopolimer yang dihasilkan mempamerkan berat molekul, sifat haba dan mekanikal
yang berjulat luas bergantung kepada komposisi monomer dan jenis substrat karbon.
xxii
BIOSYNTHESIS AND CHARACTERIZATION OF
POLY(3-HYDROXYBUTYRATE-co-4-HYDROXYBUTYRATE)
COPOLYMER FROM Cupriavidus sp. USMAHM13
ABSTRACT
Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-
4HB)] using combination of waste glycerine and carbon precursor by bacteria is still
very limited. Therefore, this research was conducted to (i) isolate and identify a
bacterium that able to produce high PHA content and 4HB monomer composition,
(ii) explore the ability of the bacterium to produce P(3HB-co-4HB) using various
inexpensive and renewable carbon sources, (iii) optimize the P(3HB-co-4HB)
copolymer production using glycerine pitch through shake-flask fermentation using
response surface methodology (RSM) and (iv) evaluate the material characteristics of
the P(3HB-co-4HB) copolymers with various 4HB monomer compositions. In this
study, a novel yellow-pigmented bacterium which exhibited ability of producing
P(3HB-co-4HB) copolymer was successfully isolated from Perak, Malaysia and
designated as Cupriavidus sp. USMAHM13. Based on the phenotypic and genotypic
analyses, it could be suggested that Cupriavidus sp. USMAHM13 represents a novel
species within the genus Cupriavidus. Preliminary screening of carbon sources for
biosynthesis of P(3HB-co-4HB) copolymer by Cupriavidus sp. USMAHM13
revealed that high 4HB monomer composition (43 mol%) with cell dry weight and
PHA content of 6.0 g/l and 49 wt%, respectively was achieved through combination
of glycerine pitch (5 g/l) and 1,4-butanediol (5 g/l) via one-stage cultivation. This
study also revealed that recovered crude glycerine from glycerine pitch contributed
the most for the synthesis of P(3HB-co-4HB) copolymer compared to the other
xxiii
recovered components. Enhancement of 4HB monomer accumulation was also
attained through the addition of ammonium acetate as nitrogen source which acted as
4HB stimulator. Medium optimization using RSM through shake-flask fermentation
had led to the highest accumulation of 4HB monomer (51 mol%) with cell dry
weight and PHA content of 10.1 g/l and 53 wt%, respectively using combination of
glycerine pitch (10 g/l), 1,4-butanediol (8.14 g/l) and ammonium acetate (2.39 g/l).
Biosynthesis of P(3HB-co-4HB) copolymer with 4HB monomer compositions
ranged from 3 mol% to 40 mol% was also achieved through batch fermentation in
bioreactor by manipulating the concentration of ammonium acetate. The P(3HB-co-
4HB) copolymers produced exhibited a wide range of molecular mass, thermal and
mechanical properties depending on the monomer compositions and type of carbon
sources.
1
1.0 INTRODUCTION
The current prominence on sustainability, eco-efficiency and green chemistry
has generated tremendous search for materials that are renewable and
environmentally friendly. Biopolymers are one of the renewable materials from
microorganisms which can provide a source of sustainable alternative to petroleum
derived plastics. A variety of biodegradable polymers such as polyhydroxyalkanoates
(PHAs), poly(ε-caprolactone) (PCL), polylactide (PLA), poly(p-dioxanone) (PPDO)
and poly(butylene succinate) (PBS) are being studied for different applications
ranging from industrial to medical applications (Akaraonye et al., 2010).
Polyhydroxyalkanoates (PHAs) are one of the versatile classes of
biodegradable polymers which constitute a group of microbial biopolyesters with
important ecosystem functions and high biotechnological potentials (Akaraonye et
al., 2010; Koller et al., 2011). It is well established that PHAs are synthesized by
bacteria and some archaea as an intracellular carbon and energy storage material
through various pathways when experience metabolic stress in the environments of
fluctuating availability and limitation of nutrient (Koller et al., 2011; Anderson and
Dawes, 1990). PHAs have evoked great interest among researchers due to their
inherent biocompatibility and biodegradability which is not surprising as monomer
of PHAs, 3-hydroxybutyric acid is a normal constituent of human blood that has
been considered in industries such as food supplement, pharmaceutical and other fine
chemicals (Ren et al., 2010).
Even though both industries and governments have increased their efforts in
the commercialization of biodegradable polymers, high production costs (4-6
USD/kg), limited microbial strains and difficulty in recovering the polymer have
hampered the widespread applications of these high-quality polymers (Akaraonye et
2
al., 2010). Development of superior PHA-producing strains and fermentation
strategies as well as the current progress in downstream process technology will
make the prices of PHA products to be competitive with their synthetic counterparts.
The isolation and development of PHA-producing microorganism that has the ability
to utilize inexpensive and renewable carbon substrates has to be pursued intensively
since half of the production cost accounts on the substrate cost (Kim, 2000; Ren et
al., 2010; Sudesh et al., 2011).
Among the diverse types of PHAs that have been revealed, copolyester
poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] has been explored
as biopolymer porous substrates in tissue engineering applications due to their
biocompatibility and desirable mechanical properties (Williams and Martin, 2002).
In fact, existence of 4-hydroxybutyric acid as normal constituent in the extracts of
brain tissue of rat, pigeon and man has classified it as one of the most valuable
biopolymer among the vast number of different PHAs synthesized by
microorganisms (Sudesh et al., 2000; Williams and Martin, 2002).
Exploring the utilization of waste materials is a good example of reducing the
substrate cost by eliminating the necessity for supplementing with the more
expensive carbon source. Recycling of wastes generated from industrial plants for
PHA production is not only crucial in improving the economics of microbial PHA
production but also for waste management (Solaiman et al., 2006; Akaraonye et al.,
2010). Currently, the rising demand for the biodiesel worldwide has led to the excess
discharge of by-product glycerine which is considered as an unrefined raw product.
This waste glycerine is the principal by-product generated during the
transesterification of vegetable oils and animal fats in the presence of catalyst.
Unrefined glycerine has become a potential environmental pollutant because
3
majority of the cosmetics, pharmaceuticals and food industries prefer purified
glycerine as a raw material. Nevertheless, glycerine purification process is an
expensive process and recently, it has become economically unfeasible due to low
prices of glycerine (Leoneti et al., 2012; da Silva et al., 2009).
It is of great importance for scientists to explore alternative potential uses of
unrefined glycerine in order to reduce its excess accumulation and to control the
economics of biodiesel production (Solaiman et al., 2006). Numerous papers have
published direct utilization of unrefined glycerine in various applications such as
feedstock in the production of different chemical products, hydrogen synthesis,
additives for automotive fuels and ethanol or methanol production. Other interesting
uses that have been considered are such as animal feed, co-digestion, co-gasification
and waste treatment. Bioconversion into high value products through microbial
fermentation is one of the most promising applications for the use of unrefined
glycerine (Yang et al., 2012).
The excess production of waste glycerine is also creating problems for
oleochemicals industries due to the collapse in crude glycerine prices which have
fallen from about $0.25 per pound to $0.05 per pound. The producers need to pay to
remove the crude glycerine from their plants and incinerate it. One of the US
government agencies, Department of Energy has adopted the promotion of new
glycerine platform chemistry and product families as one their most important goals
to meet the need for obtaining new chemicals (Yang et al., 2012; Dharmadi et al.,
2006).
Oleochemicals industry in Malaysia has been diversifying significantly due to
the plentiful supply of kernel and palm oils as raw materials as well as the high
demand for downstream products such as glycerine, fatty alcohols and fatty acids.
4
However, environmental awareness is growing rapidly in Malaysia because
oleochemicals industry is one of the palm-oil based industries that possess risk to the
environment. Approximately, 494 kg of glycerine pitch is generated daily in
Malaysia and it is treated and disposed at prescribed premises. The cost for landfill is
~163 US$ whereas for incineration is ~260 US$ - 1172 US$ per tonne (Hazimah et
al., 2003; Hidawati and Sakinah, 2011).
1.1 Problem statements
About two million species of microbes has been estimated in Malaysia as a
major resource for innovative biotechnological products processes. However,
microbial diversity remains an unexploited resource as only about 17% of the total
number of estimated numbers of bacteria and fungi have been reported by the year
2000 (Vikineswary, 1998; Krishnapillay et al., 2003). At present, only two bacteria
that capable of producing P(3HB-co-4HB) copolymer with various 4HB monomer
compositions have been isolated from Malaysian environment. The bacteria are
Cupriavidus sp. USMAA1020 and Cupriavidus sp. USMAA2-4 that isolated from
Lake Kulim, Kedah and Sg. Pinang, Penang, respectively (Amirul et al., 2008; Chai
et al., 2009). Therefore, the search for new P(3HB-co-4HB)-producing bacterial
strains from Malaysian environment still remains of interest as Malaysia is one of the
world’s twelve mega diversity areas with exceptionally rich biological resources.
Disposal of combustible wastes like glycerine pitch has been a major problem
to the community. Burning the waste can literally mean converting it into acrolein, a
highly volatile compound and well-known for its toxicity and very hazardous to life
(Hazimah et al., 2003). Biological conversion of glycerine pitch as potential carbon
substrate into microbial polyester would give positive impact on both economic and
environmental aspect. Production of P(3HB-co-4HB) copolyester using combination
5
of waste glycerine with addition of carbon precursor are still limited. It is imperative
to study the variable factors affecting the P(3HB-co-4HB) copolyester accumulation
using glycerine pitch and to systematically monitor the compositions of waste
glycerine as such information could help to define acceptable range for feedstock
variability of this waste carbon.
1.2 Objectives
In the present study, bioprospection of P(3HB-co-4HB)-accumulating
bacteria was performed from Malaysian environment. The potential bacterium was
selected based on its ability to convert carbon precursor into high 4HB monomer
composition. The isolated bacterium was identified and characterized based on
physiological and molecular analysis. Production of P(3HB-co-4HB) copolymer by
the isolated bacterium using glycerine pitch was focused throughout the study.
The objectives of this study were;
1. To isolate and identify a bacterial strain with ability to produce high PHA content
and 4HB monomer composition
2. To explore the ability of the newly isolated strain to produce P(3HB-co-4HB)
using various inexpensive and renewable carbon sources
3. To optimize the P(3HB-co-4HB) copolymer production using glycerine pitch
through shake-flask fermentation using response surface methodology (RSM)
4. To evaluate the material characteristics of the P(3HB-co-4HB) copolymers with
various 4HB monomer compositions
6
2.0 LITERATURE REVIEW
2.1 Polyhydroxyalkanoates: The prospect green biopolymer
Polyhydroxyalkanoates (PHAs) are one of the greatly fascinating families of
microbial polyesters of 3, 4, 5 and 6 hydroxyacids that have promising potentials in
various industrial and medical applications due to their wide range of characteristics
(Akaraonye et al., 2010; Philip et al., 2007). These polymers with imperative
ecosystem roles and high biotechnological potentials are synthesized naturally by a
diverse range of bacterial species from at least 75 different genera (Koller et al.,
2011; Reddy et al., 2003). The polymers are usually accumulated as insoluble
inclusions in the cytoplasm of bacterial cells during the depletion of essential
nutrients such as nitrogen, magnesium or phosphorus in the presence of abundant
carbon sources (Tian et al., 2009).
Most of the bacteria accumulate these polymers as storage materials in the
form of mobile, amorphous, liquid granules for their survival under stress or hostile
conditions (Luengo et al., 2003). PHAs also serve as a sink for reducing equivalents
for some microorganisms. The insolubility of PHAs inside the bacterial cytoplasm
causes insignificant increase in the osmotic pressure, thus preventing the leakage of
these valuable compounds out of the cells while securing the stored nutrient at a low
maintenance cost (Rehm, 2006; Verlinden et al., 2007).
PHAs exhibit thermoplastic and elastomeric properties after they are
extracted from the cells. These biopolymers are the only waterproof thermoplastic
materials available that are fully biodegraded in the terrestrial and aquatic
ecosystems by microorganisms through both aerobic and anaerobic conditions
(Philip et al., 2007; Lee, 1996). PHAs can be used in various ways similar to many
7
non-biodegradable synthetic plastics by varying their toughness and flexibility,
depending on their formulations (Verlinden et al., 2007).
In addition, PHAs have caught the attention of many researchers due to their
inherent biocompatibility. The medically attractive characteristics of these
biopolymers have become the focus of many investigations. PHAs are more
favourable for the development of tissue-engineered scaffold because they have been
proven biocompatible in tissue engineering. They also exhibit medically important
characteristics that are not found in the present synthetic absorbable polymer such as
polyglycolic acid (PGA). Hence, these polymers serve as excellent substitute for
synthetic plastics due to their ease of processability, tailor-made physical
characteristics, biodegradability and biocompatibility (Sudesh et al., 2000; Valappil
et al., 2006).
The incorporated 3-hydroxyalkanoic monomers units of the PHAs are all in
the R(–) configuration due to the stereospecificity of the polymerizing enzyme, PHA
synthase. Therefore, PHAs containing R(–) 3HA monomers units represent a family
of the optically active microbial polyesters. Small portion of S monomers are
detected only in unusual cases. Biosynthesis of PHAs by bacteria will warrant the
incorporation of R(–) HA monomers which is indispensable for the biodegradability
and biocompatibility of these polymers (Sudesh et al., 2000; Akaraonye et al., 2010;
Zinn and Hany, 2005). Hydrolysis of PHAs will produce R-hydroxyalkanoic acids
that can be used as chiral starting materials in fine chemicals, pharmaceutical and
medical industries (Philip et al., 2007).
The material properties of the polymers such as melting temperature, glass
transition temperature and crystallinity are greatly influenced by the length of the
side chain and the functional group of polymers. Most of the PHAs exhibit thermal
8
and mechanical properties that are comparable to petroleum-based plastics, such as
polypropylene. The variable properties of the polymer will determine the final
application of these polymers in various industrial and medical fields (Akaraonye et
al., 2010; Brigham and Sinskey, 2012).
2.1.1 History of PHAs
In 1963, Chowdhury reported that the PHA granules which occur as refractile
bodies in the bacterial cells were first observed under the microscope by Beijerinck
in 1888. However, only in 1927, the composition of PHAs was firstly reported by the
French scientist, Maurice Lemoigne. Inclusion bodies found in Bacillus megaterium
that primarily consists of poly(3-hydroxybutyrate) [P(3HB)] were characterized by
Lemoigne who worked at the Lille branch of the Pasteur Institute - France. Lemoigne
was the first to report that the bacterial granules are not ether soluble as in lipids and
act as reserve material components (Braunegg et al., 1998; Amara, 2008).
In 1974, Wallen and Rohwedder reported the presence of 3-hydroxybutyrate
(3HB) and 3-hydroxyvalerate (3HV) as major monomers with C6 and probably C7
as minor components from the activated sewage sludge that extracted using
chloroform. This heteropolymer showed distinguishable properties with P(3HB) as it
exhibited lower melting temperature and was soluble in hot ethanol. This was the
first report on the presence of other 3-hydroxyacids than 3HB (Anderson and Dawes,
1990).
De Smet et al. (1983) reported significant progress whereby Pseudomonas
oleovorans was found to accumulate 3-hydroxyoctanoate (3HO) and an unidentified
fatty acid when grown on n-octane (50%, v/v). Subsequently, a more detailed
investigation by Lageveen et al. (1988) disclosed that the unidentified fatty acid
9
accumulated by Pseudomonas oleovorans was (R)-3-hydroxyhexanoate (3HHx).
P(3HB) is not synthesized by Pseudomonas oleovorans from either n-alkanes or
glucose.
At least 11 short-chain 3-HAs with 3HB and 3HV being the major
components were detected using gas chromatography analysis in the polymer
extracted from marine sediments. Presence of 95% 3HB, 3% 3-hydroxyheptanoate
(3HHp), 2% 3HO and trace amounts of three other 3-HAs were detected in the
purified polymer extracted from Bacillus megaterium (Findlay and White, 1983).
This was followed by the discovery of PHAs containing C4, C6 and C8 monomers
from sewage sludge (Odham et al., 1986).
The production of P(3HB) was fully developed on the industrial scale only in
the early 1960s. Several patents were obtained by Baptist and Werber at W.R. Grace
& Co. (U.S.A) for their pioneering works related to P(3HB) production by
fermentation and fabrication of absorbable prosthetic devices. Tremendous increase
in the search for alternative plastics was boosted by the oil crisis in 1970. This
opportunity was taken by the Imperial Chemical Industries (ICI) from United
Kingdom to formulate conditions that able to produce 70 wt% of P(3HB)
homopolymer using Alcaligenes latus. A novel P(3HB-co-3HV) copolymer under
trademark BIOPOL® was also produced by ICI. In April 1996, a range of P(3HB-co-
3HV) marketed under the trademark BIOPOL® was produced using Cupriavidus
necator by Monsanto which purchased the BIOPOL® business from Zeneca Bio
(branch of ICI). In 1998, Metabolix Inc. obtained the licence from Monsanto and
launched a new spin off company named Tepha following the alliance between
Metabolix Inc. and Children’s Hospital, Boston. Tepha, Inc. is a medical device
company headquartered in Lexington, Masachussetts, US which develops innovative
10
medical devices based on PHA polymers such as P(4HB) homopolymer through
advancement of biotechnology and material sciences to be used in procedures for
surgical repair and regenerative medicine (Philip et al., 2007; Braunegg et al., 1998).
2.1.2 Types of PHAs and their physical properties
Although PHAs are considered consumer-oriented and environmentally
friendly biopolymer due to their biodegradability and biocompatibility,
commercialization of these biopolymers is stringently dependent on the material
properties that satisfy the requirement of the targeted market application. At present,
more than 200 different monomer constituents are found either as homopolyester or
in combination as copolyester (Gomez et al., 2012). Wide substrate range of the
PHA synthase has resulted in the versatility of the monomer compositions which is a
clear advantage because the monomer variation provides PHAs an extended
spectrum of associated properties. PHAs are classified into three classes according to
their monomer compositions; Short-chain-length (SCL)-PHAs, medium-chain-length
(MCL)-PHAs and short-chain-length-medium-chain-length (SCL-MCL)-PHAs
(Steinbüchel and Lütke-Eversloh, 2003; Chen, 2009).
2.1.2.1 Short-chain-length (SCL)-PHAs
SCL-PHAs are polymers of 3-HA monomers with a chain length of three to
five carbon atoms. They are stiff materials that have methyl and ethyl groups as
small side chains. These polymers exhibit high tensile strength and crystallinity but
with low elongation to break depending on their monomer compositions (Doi et al.,
1995). Homopolymer P(3HB) is the most well-known microbial polyester produced
by wide ranges of microorganisms and has comparable material properties with
polypropylene (Anderson and Dawes, 1990). Nevertheless, P(3HB) is a rigid and
11
brittle polymer with low elasticity, thus making it unfavourable for industrial use due
to its limited applications. The brittleness of P(3HB) is due to its perfect
stereoregularity which requires it to undergo a detrimental aging process at ambient
temperatures (de Koning and Lemstra, 1993). It is also difficult to process this
homopolymer because it exhibits high melting temperature of 170°C (Sudesh et al.,
2000).
Manipulating the side chains and compositions of P(3HB) polymers through
incorporation of other monomers can generate different types of polymers with
favourable material properties as the polymers will confer less stiffness and tougher
properties. Introducing the methyl and ethyl groups as side chains into the polyster
backbone can improve the ductility of P(3HB) by disturbing or reducing the crystal
lattice of P(3HB) (Doi et al., 1995). Among the short-chain-length polymers that
have been studied with such material properties are copolymers poly(3-
hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)], poly(3-hydroxybutyrate-
co-4-hydroxybutyrate) [P(3HB-co-4HB)] and terpolymer poly(3-hydroxybutyrate-
co-3-hydroxyvalerate-co-4-hydroxybutyrate) [P(3HB-co-3HV-co-4HB)] (Park et al.,
2012).
P(3HB-co-3HV) copolymer is one of the most well-characterized polyester
that has attracted industrial attention (Bhubalan et al., 2008). Incorporation of 3HV
monomers into the 3HB monomer chains will increase the Young's modulus,
elasticity, tensile strength and toughness of the P(3HB-co-3HV) copolymer (Madden
et al., 2000; Ojumu et al., 2004). Decrease in the melting temperature with
incorporation of 3HV monomer has allowed better thermal processing of P(3HB-co-
3HV) copolymer and even better biodegradation process because decrease in the
12
melting temperature is coupled without any changes in the degradation temperature
(Bluhm et al., 1986).
P(3HB-co-4HB) copolymer is a very promising but insufficiently studied
PHA. This copolymer exhibits good biocompatibility, resorbability, elastomeric
properties and are biodegraded in vivo and in the environment at high rates
(Vigneswari et al., 2012; Zhila et al., 2011). Fabrication of P(3HB-co-3HV-co-4HB)
terpolymer is initiated for producing a hybrid polymer possessing the superior and
desirable physical and mechanical properties of both P(3HB-co-3HV) and P(3HB-
co-4HB) copolymers. According to Aziz et al. (2012), enhancement of the
mechanical and physical properties of the terpolymer P(3HB-co-3HV-co-4HB) can
be achieved by incorporating different proportions of both 3HV and 4HB monomer
units into the terpolymer chain. Terpolymers with superior material properties are
desirable in the medical and pharmaceutical fields. Ramachandran et al. (2011) has
reported production of terpolymer P(63%3HB-co-4%3HV-co-33%4HB) with high
Young's modulus (101 MPa) and elongation to break (937%) which is suitable for
medical applications such as sutures, cardiovascular stents and vascular grafts.
2.1.2.2 Medium-chain-length (MCL)-PHAs
MCL-PHAs consist of monomers with 6 to 14 carbon atoms. The first
discovery of MCL-PHAs is a polyester containing 3-hydroxyoctanoic acids (3HO)
synthesized by Pseudomonas oleovorans (Steinbüchel and Lütke-Eversloh, 2003).
Typical MCL-PHAs are poly(3-hydroxyhexanoate-co-3-hydroxyoctanoate-co-3-
hydroxydecanoate) [P(3HHx-co-3HO-co-3HD)] and poly(3-hydroxyhexanoate-co-3-
hydroxyoctanoate-co-3-hydroxydecanoate-co-3-hydroxydodecanoate) [P(3HHx-co-
3HO-co-3HD-co-3HDD) (Chen, 2010). These PHAs are usually represented by the
13
most versatile PHA accumulators, Pseudomonads which belong to the rRNA-
homology-group I. This group of bacteria derives the 3-hydroxyacyl-CoA from the
intermediates of fatty acid β-oxidation pathway for the polymerization reaction by
MCL-PHA synthase when they are grown on aliphatic alkanes or fatty acids (Sudesh
et al., 2000).
The monomer composition of MCL-PHAs produced is usually related to the
substrate used with most units have 2 carbon atoms lesser than the provided carbon
source (Ojumu et al., 2004; Braunegg et al., 1998). Enoyl-CoA hydratase (PhaJ) and
3-ketoacyl-CoA reductase (FabG) are the specific enzymes that involved in the
conversion of intermediates of fatty acid β-oxidation into the suitable monomers used
in the PHA polymerization by MCL-PHA synthase. Copolyester consisting of (R)-
3HO as main monomer and (R)-3HHx as minor monomer is accumulated by
Pseudomonas putida cultivated on octanoic acid as carbon source (Steinbüchel and
Lütke-Eversloh, 2003).
MCL-PHAs can also be synthesized by most of the Pseudomonas sp. using
structurally unrelated carbon sources such as gluconate, fructose, acetate, glycerine
and lactate. The precursors for these polymers are provided by de novo fatty acid
synthesis and converted from acyl carrier protein (ACP) form to CoA through
catalytic reaction by 3-hydroxyacyl-CoA-ACP transferase (PhaG) (Yu, 2007).
Monomers for MCL-PHAs biosynthesis are also generated by malonyl-CoA-ACP
transacylase (FabD) which is an over-expressed transacylating enzyme. P.
aeruginosa, P. aureofaciens, P. citronellolis, P. mendocina and P. putida are among
the Pseudomonads that have been revealed to synthesize MCL-PHAs through this
pathway (Sudesh et al., 2000). Production of MCL-PHAs comprising of 7 different
monomers; 3HD (major constituent), 3HHx, 3HO, saturated and mono-unsaturated
14
monomers of 12 and 14 carbon atoms by P. putida grown on glucose has been
reported by Huijberts and Eggink (1996).
2.1.2.3 Short-chain-length-medium-chain-length (SCL-MCL)-PHAs
According to Chen (2009), copolyesters of SCL and MCL monomers are the
ideal biomaterials for the advancement of various applications because they exhibit
useful and flexible mechanical properties. MCL-PHAs are more elastomer in nature
compared to SCL-PHAs which are often stiff and brittle. Incorporating both
monomers will result in SCL-MCL PHA copolymers exhibiting properties between
the two states which will depend on the different proportions of SCL and MCL
monomers. This copolymer has superior properties compared to the SCL and MCL
homopolymer. Therefore, it is desirable to elucidate new and low cost ways to
synthesize SCL-MCL-PHAs with a small molar fraction of MCL monomers from
renewable resources (Nomura et al., 2004).
P(3HB-co-3HHx) copolymer is one of the successful SCL-MCL-PHAs that is
produced on an industrial scale (Chen, 2009). High yield production of P(3HB-co-
3HHx) copolymer has been obtained successfully using renewable soybean oil by
Cupriavidus necator and its recombinant (Kahar et al., 2004). Bhubalan et al. (2008)
has proven the good choice of palm kernel oil as the primary carbon source together
with the addition of sodium propionate and sodium valerate as 3HV carbon
precursors for the production of P(3HB-co-3HV-co-3HHx) terpolymers having novel
compositions with attractive properties. SCL-MCL-PHA copolymers comprising C4
and C6-C12 have been trademarked as NodaxTM by US-based Procter & Gamble
(Noda et al., 2010). Table 2.1 shows the chemical structure of common PHAs.
15
Table 2.1: The chemical structure of common PHAs (Braunegg et al., 1998; Brigham and Sinskey, 2012)
Chemical structure Polymer
CH3 O P(3HB)
[-O-CH-CH2-C-]
CH3 P(3HB-co-3HV)
CH3 O CH2 O
[-O-CH-CH2-C-]-[-O-CH-CH2-C-]
P(3HB-co-4HB) CH3 O O
[-O-CH-CH2-C-]-[O-CH2-CH2-CH2-C-]
CH3 P(3HB-co-3HHx)
CH2
CH3 O CH2 O
[-O-CH-CH2-C-]-[-O-CH-CH2-C-]
CH3 P(3HB-co-3HV-co-4HB)
CH3 O CH2 O O
[-O-CH-CH2-C-]-[-O-CH-CH2-C-]-[O-CH2-CH2-CH2-C-]
CH3 P(3HB-co-3HV-co-3HHx)
CH3 CH2
CH3 O CH2 O CH2 O
[-O-CH-CH2-C-]-[-O-CH-CH2-C-]-[-O-CH-CH2-C-]
16
2.1.3 Biosynthesis of PHAs
2.1.3.1 Biosynthesis of SCL-PHAs
In 1969, Richie and Dawes revealed the involvement of an acyl carrier
protein (ACP) and CoA esters as the intermediates in the PHA synthesis (Dawes,
1988). Three main ezymes involved in the biosynthesis route from acetyl-CoA are;
3-ketothiolase, acetoacetyl-CoA reductase and PHA synthase. The controlling
enzyme in the PHA biosynthesis is 3-ketothiolase with CoA as key effector
metabolite. Two molecules of acetyl-CoA are coupled in a condensation reaction by
3-ketothiolase (PhaA) to generate acetoacetyl-CoA through the release of the CoA
(Anderson and Dawes, 1990). This reversible reaction catalyzed by 3-ketothiolase
that inhibited by the presence of excess free CoA is discovered by Senior and Dawes
(1973). Subsequently, the acetoacetyl-CoA is stereoselectively reduced to R(–)-3-
hydroxybutyryl-CoA by acetoacyl-CoA reductase (PhaB). Two acetoacetyl-CoA
reductases (NADH and NADPH) possessing different substrate and coenzyme
specificities have been found in Cupriavidus necator. Since PHA synthase of
Cupriavidus necator is specific for R(–)-substrates, only the NADPH reductase
involved in the PHA synthesis from acetyl-CoA (Kessler and Witholt, 2001).
According to Dawes (1988), the acetoacetyl-CoA reductase, a typical thiol enzyme is
five times more active with NADPH than NADH.
PHA synthase (PhaC) polymerizes the monomers with the release of CoA.
This enzyme which is bound with the membrane of the PHA granules, determines
the type of PHAs synthesized by bacteria. PHA synthase is distinguished into three
types based on the substrate specificities and primary structures. The active site of
the PHA synthase that takes part in the polymerization process is a strictly conserved
17
cysteine residue (Sudesh et al., 2000). PHA synthase of Cupriavidus necator is
active with C4 and C5 substrate and also specific for R(–) enantiomers. This
indicates active preferences of the PHA synthase of Cupriavidus necator towards
SCL monomers. Since the position of the oxidized carbon in the PHA monomers is
usually not a vital factor, this enzyme can incorporate 4-HA and 5-HA besides the
common 3-HA (Anderson and Dawes, 1990; Sudesh et al., 2000). Polymerization
involves the reaction of soluble components of the cytoplasm at a surface to form a
hydrophobic product which apparently accumulates in a very hydrophobic
environment within the granules through a two-stage process reaction. This reaction
involves the generation of an acyl-enzyme intermediate through a functional thiol
group on the enzyme (Dawes, 1988).
2.1.3.2 Biosynthesis of MCL-PHAs
Biosynthesis of MCL-PHAs consisting of (R)-3-hydroxy fatty acids is
performed through conversion of fatty acid metabolism intermediates to the (R)-3-
hydroxyacyl-CoA. Conversion of fatty acid β-oxidation intermediates into suitable
monomers for the polymerization process by the PHA synthase requires the
involvement of specific enzymes, enoyl-CoA hydratase (PhaJ) and 3-ketoacyl-CoA
reductase (FabG) (Rehm, 2006; Sudesh et al., 2000). The (R)-specific hydration of 2-
enoyl-CoA is catalyzed by the (R)-specific enoyl-CoA hydratase (PhaJ) to supply the
(R)-3-hydroxyacyl-CoA monomer which is the substrate for the polyester synthase
(PhaC) (Fukui et al., 1998).
Biosynthesis of de novo fatty acid intermediates which exclude the fatty acid
β-oxidation pathway are the alternative route for MCL-PHA synthesis in the bacteria.
This pathway is the main route employed by the bacteria cultivated on unrelated
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carbon sources such as carbohydrate, acetate or ethanol to synthesize 3-hydroxyacyl-
CoA (Suriyamongkol et al., 2007). In this pathway (FabD pathway), the conversion
of the (R)-3-hydroxyacyl moiety of the respective ACP (acyl carrier protein)
thioester to its corresponding CoA thioester is catalyzed by the malonyl-CoA-ACP
transacylase (FabD). 3-hydroxyacyl-ACP-CoA transferase (PhaG) also involves in
the conversion of (R)-3-hydroxyacyl-ACP to (R)-3-hydroxyacyl-CoA which is a
substrate for PHA synthase (Yu, 2007; Sudesh et al., 2000). Figure 2.1 illustrates the
biosynthetic pathways involve in synthesizing various types of PHAs.
Figure 2.1: Biosynthetic pathways of short-chain-length (SCL)-PHA, medium-chain- length (MCL)-PHA and short-medium-chain-length (SCL-MCL)-PHA from carbohydrates. PhaA, β-ketothiolase; PhaB, NADPH-dependent acetoacetyl-CoA reductase; PhaC, PHA synthase; PhaG, 3-hydroxyacyl-ACP-CoA transferase; PhaJ, (R)-specific enoyl-CoA hydratase. Dotted lines represent reactions where intermediate metabolic steps are not included (Aldor and Keasling, 2003; Sudesh et al., 2000).
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2.2 Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)]
2.2.1 Biosynthesis of P(3HB-co-4HB) copolymer by various microorganisms
A very promising and interesting candidates for biomaterial is the P(3HB-co-
4HB) copolymer due to the existence of 4-hydroxybutyrate monomer that reduces
the crystallinity of polymer but enhances the polymer’s flexibility characteristic.
(Chanprateep et al., 2010; Zhila et al., 2011). Production of PHAs consisting of 4HB
monomer by various microorganisms has been investigated since early 1990s. Wild-
type strains capable of biosynthesizing P(3HB-co-4HB) copolymer from different
carbon sources are Cupriavidus necator (Doi, 1990; Nakamura and Doi, 1992;
Valentin et al., 1995; Lee et al., 2000; Kim et al., 2005; Chanprateep et al., 2008;
Chanprateep et al., 2010; Rao et al., 2010; Saito et al., 1996; Volova et al., 2011),
Alcaligenes latus (Hiramitsu et al., 1993; Saito et al., 1996; Kang et al., 1995),
Comamonas testosteronii (Renner et al., 1996), Delftia acidovorans (Kimura et al.,
1992; Saito et al., 1996; Sudesh et al., 1999; Lee et al., 2004; Mothes and
Ackermann, 2005; Hsieh et al., 2009; Ch’ng et al., 2012), Hydrogenophaga
pseudoflava (Choi et al., 1999) and Chromobacterium sp. (Zhila et al., 2011).
Saito et al. (1996) reported similar observation as made by Kunioka Masao in
1988 who demonstrated production of random copolymer of P(3HB-co-4HB) by
Cupriavidus necator using γ-butyrolactone, 4-hydroxybutyric acid and alkanediols of
even number (1,4-butanediol, 1,6-hexanediol, 1,8-octanediol, 1,10-decanediol and
1,12-dodecanediol). Molar fraction of 4HB ranging from 9 mol% to 34 mol% was
produced using various carbon sources. Decrease in the 4HB molar fraction was
observed when fructose or butyric acid was added into the nitrogen-deficient medium
containing 4-hydroxybutyric acid or γ-butyrolactone. Similar synthesis of P(3HB-co-
20
4HB) copolymer using various carbons was also carried out using Delftia
acidovorans DS-17 that isolated from activated sludge. This bacterium did not
accumulate 3HB monomer when grown on 1,4-butanediol or 4-hydroxybutyric acid
which proposes the restriction of 4-hydroxybutyryl-CoA metabolism to acetyl-CoA.
Inability of this bacterium to metabolize 4-hydroxybutyryl-CoA into (R)-3-
hydroxybutyryl-CoA had resulted in the synthesis of P(4HB) homopolymer with
PHA content ranged from 21 wt% to 28 wt%.
According to Kim et al. (2005), bacterial growth was inhibited by high
concentration of fructose (> 20 g/l) and γ-butyrolactone (> 6 g/l) in the biosynthesis
of P(3HB-co-4HB) copolymer by Cupriavidus necator, suggesting that a controlled
feeding rate of fructose and γ-butyrolactone should be employed as one of the
strategies in the fed-batch fermentation. Acetate as well as propionate were also used
as stimulator at concentration of 2 g/l to increase the 4HB monomer incorporation
from 38 mol% to 54 mol%.
High proportions of 4HB unit (60 mol%-100 mol%) was also produced by
Cupriavidus necator using 4-hydroxybutyric acid supplemented with additives such
as ammonium sulphate and potassium dihydrogen citrate, however the polyester
content was found to decrease (Saito et al., 1996). Regulation of 4HB molar fraction
through supplementation of propionate was also reported by Lee et al. (2000),
suggesting that increment of 4HB monomer composition from 12 mol% to 52 mol%
through addition of propionate in small amount together with γ-butyrolactone was
due to the inhibition of ketolysis reaction which catalyzes the lysis of 4HB-CoA to
two units of acetyl-CoA.
Chanprateep et al. (2008) demonstrated efficient accumulation of 4HB
monomer by newly isolated Cupriavidus necator strain A-04 through shake-flask
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fed-batch cultivation that was influenced by the carbon precursors such as γ-
hydroxybutyric acid and 1,4-butanediol. It was suggested that monomer
compositions of 4HB could be regulated from 0 mol% to 70 mol% by manipulating
the concentrations of two carbon substrates and the carbon-to-nitrogen ratio. Specific
production rate of 3HB monomer was the highest at C/N 200 whereas maximum
specific production rate of 4HB monomer was attained when C/N ratio used is
between 4 and 20.
The feasibility production of P(3HB-co-4HB) copolymer by Cupriavidus
necator using the spent palm oil left after frying activities and 1,4-butanediol was
demonstrated by Rao et al. (2010) who reported high PHA yield (0.75–0.8 g/g of
spent palm oil) with constant accumulation of 4HB monomer (15 mol%) that led to
a conclusion that 4HB monomer accumulation was not influenced by the cultivation
period and the existence of polar solids in the spent palm oil.
Cavalheiro et al. (2012) presented the first report on the production of
P(3HB-co-4HB) copolymer from waste glycerine using high-cell density fed-batch
cultures of Cupriavidus necator DSM 545. Incorporation of 4HB monomers was
initiated by adding γ-butyrolactone. P(3HB-co-4HB) copolymers with 11 mol% to
22 mol% of 4HB monomer were attained by manipulating the dissolved oxygen
concentration and cultivation time. Monomer of 4HB was increased by 2-fold using
propionic acid as a stimulator but it had resulted in the formation of P(3HB-co-3HV-
co-4HB) terpolymer because propionic is a precursor for formation of HV monomer.
Delftia acidovorans possesses the most efficient metabolic pathway for the
biosynthesis of P(3HB-co-4HB) copolymer and generally, PHAs extracted from this
bacterium are safe as tested based on cytotoxicity, genotoxicity and implant tests.
Even though Delftia acidovorans is a potential strain for the production of P(3HB-
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co-4HB) copolymer for medical applications, but it has inferior ability of controlling
the monomer compositions in a wide range (Ch’ng et al., 2012; Siew et al., 2009).
According to the study carried out by Hsieh et al. (2009), P(3HB-co-94%4HB)
copolymer was achieved with cell concentration and PHA content of 2.5 g/l and 13
wt%, respectively using Delftia acidovorans cultivated under optimal conditions as
follows; 1,4-butanediol (10 g/l), pH (7), incubation time (72 hours) and temperature
(26ºC). Delftia acidovorans was also reported to produce P(3HB-co-4HB)
copolymers with extremely high 4HB compositions (93-99 mol%) with PHA
content of 12 wt% to 18 wt% using 1,4-butanediol (5 g/l) as sole carbon source
through two-stage cultivation process (Kimura et al., 1992).
According to Lee et al. (2004), besides adjusting the concentration of carbon,
increase in 4HB monomer composition accumulation by Delftia acidovorans could
also be achieved by adjusting pH and aeration. It was reported that influence of pH
was greater on the generation/incorporation of 3HB monomers rather than on 4HB
monomers directly, leading to the suggestion that pH affected the intracellular
concentration of acetyl-CoA. The authors also reported that molar fraction of 4HB
could be significantly enhanced without adversely affecting the PHA content by
reducing the aeration as the conditions prevented the incorporation of 3HB
monomer.
Effect of Mg2+ concentrations on the compositions of P(3HB-co-4HB)
copolymer produced by Delftia acidovorans using glucose and 1,4-butanediol had
been reported by Lee et al. (2007). The authors demonstrated that 3HB monomer
decreased with the increase of Mg2+ which was due to the decrease in the uptake of
glucose by the bacteria as the stability of membrane was disrupted by the ionic
interactions with the phosphonyl group. Concentration of Mg2+ influences the
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transport of glucose across the membrane which eventually affects the generation of
acetyl-CoA required to synthesize the 3HB monomer. P(3HB-co-4HB) copolymer
content was also very low in the absence of Mg2+, suggesting that enzymes involve
in the conversion of glucose and 1,4-butanediol to 3HB and 4HB monomer require
Mg2+ as a cofactor which will bind to the substrate to orient them properly for the
reaction.
Choi et al. (1999) reported that 4HB contents up to 66 mol% could be
produced by Hydrogenophaga pseudoflava cultivated using combination of glucose
and γ-butyrolactone through one-step cultivation process. Higher 4HB monomer
compositions (89-95 mol%) was achieved through two-step cultivation processes.
Random P(3HB-co-4HB) copolymers having broad range of 4HB monomer
compositions from 0 mol% to 83 mol% were produced by Alcaligenes latus using
combination of 3-hydroxybutyric and 4-hydroxybutyric acids (Kang et al., 1995).
Bacterial strain, Comamonas testosteronii had been investigated also for its
potential of producing P(3HB-co-4HB) copolymer using various carbon sources and
precursors which yielded molar fraction of 4HB monomer above 90 mol%. The
remarkably high 4HB monomer composition accumulated by this strain was
attributed to the very low degradation of 4HB-CoA into 3HB-CoA via acetyl-CoA.
Although high concentration of 1,4-butanediol presents in the medium, the bacterium
only utilized the stored PHA as main carbon and energy source when the acetate as
carbon source became limited. The inability to metabolize 4-hydroxybutyric acid as
carbon and energy source seems to be the reason for the accumulation of high 4HB
monomer by Comamonas testosteronii (Renner et al., 1996).
In recent years, wild-type strains of Cupriavidus sp. USMAA1020 (Amirul et
al., 2008; Amirul et al., 2009; Vigneswari et al., 2009; Vigneswari et al., 2010) and
24
Cupriavidus sp. USMAA2-4 (Chai et al., 2009; Rahayu et al., 2008) and
recombinant strains of Cupriavidus necator, Escherichia coli, Aeromonas hydrophila
and Pseudomonas putida (Zhang et al., 2009; Valentin and Dennis, 1997; Li et al.,
2010) are described as competent P(3HB-co-4HB) producers.
Cupriavidus sp. USMAA1020 and Cupriavidus sp. USMAA2-4 were isolated
from Malaysian environment. Both strains have the ability to produce P(3HB-co-
4HB) copolymer with wide ranges of 4HB monomer compositions through one-stage
and two-stage cultivation processes using various carbon precursors (Amirul et al.,
2008; Chai et al., 2009). High 4HB monomer composition of 99 mol% with PHA
content of 28 wt% was attained using Cupriavidus sp. USMAA2-4 grown in medium
containing 1,6-hexanediol through two-stage cultivation stage (Chai et al., 2009).
P(3HB-co-27%4HB) of 44 wt% content was produced by Cupriavidus sp.
USMAA2-4 cultivated for 60 hours through one-stage cultivation using combination
of oleic acid (0.5 wt% C) and 1,4-butanediol (0.5 wt% C). Different yields of
P(3HB-co-4HB) contents ranging from 47 wt% to 58 wt% were obtained by
employing new strategy of adding oleic acid and 1,4-butanediol with different
concentrations together and separately. Higher PHA content of 58 wt% was obtained
by adding 1,4-butanediol into medium containing oleic acid after 48 hours of
cultivation (Rahayu et al., 2008).
Cupriavidus sp. USMAA1020 was able to produce 4HB molar fractions
ranged from 6 to 14 mol% with high PHA content of 47 wt% to 60 wt% using γ-
butyrolactone when C/N ratio was increased from 10 to 60 through one-stage
cultivation. Higher 4HB molar fraction of 60 mol% was achieved using γ-
butyrolactone (20 g/l) through two-stage cultivation (Amirul et al., 2008). Effect of
culture conditions such as phosphate ratio, cell concentration and aeration on the
